Understanding how axons find their synaptic targets is a fundamental problem in neurodevelopment. While great progress has been made in elucidating the mechanisms of axon guidance, less is known about how axons fasciculate within specific regions of a nerve bundle. We propose to study this problem in C. elegans with a focus on the HSN axon. The HSN axon extends along the ventral nerve cord after the entire nervous system has been established. The HSN growth cone fasciculates along the PVQ and PVP axons, and the short-term goal of this project is to identify the cues that define the HSN's ability to grow along its specific path in the nerve bundle. While little is known about how axons fasciculate within mammalian nerves, recent findings suggest that sorting of olfactory axons within the nerve are important in their targeting at the olfactory bulb. The HSN may provide a model for how such nerves are organized in mammals. The proposal has three specific aims. In the first aim, the interface between the different axons will be labeled using GRASP. In the second aim, RNAi approaches to identify genes involved in HSN axon fasciculation will be developed. These approaches should bypass the lethal effects caused by RNAi of essential genes and indirect effects caused by earlier defects in the fasciculation of other axons. In the third aim, the approaches will be tested to elucidate the role of the neurotransmitter acetylcholine in axon fasciculation.. PUBLIC HEALTH RELEVANCE: Understanding how these axons find their targets is a fundamental problem in neurodevelopment. While great progress has been made in elucidating the mechanisms of axon guidance, less is known about how axons grow along specific regions of a nerve bundle. We propose to study this problem in C. elegans, a model organism that has been instrumental in defined conserved molecules that function axon guidance.